INHIBITORY MECHANISM OF SERPINS - LOOP INSERTION FORCES ACYLATION OF PLASMINOGEN-ACTIVATOR BY PLASMINOGEN-ACTIVATOR INHIBITOR-1

Serpin inhibitors are believed to form an acyl enzyme intermediate wit h their target proteinases which is stabilized through insertion of th e enzyme-linked part of the reactive center loop (RCL) as strand 4 in beta-sheet A of the inhibitor. To test critically the role and timing of these steps in the reaction of the plasminogen activator inhibitor PAI-1, we blocked the vacant position 4 in beta-sheet A of this serpin with an octapeptide. The peptide-blocked PAI-1 was a substrate for bo th tissue-type plasminogen activator (tPA) and trypsin and was hydroly zed at the scissile bond. The reactivity of the peptide-blocked substr ate PAI-1 was compared to that of the unmodified inhibitor by rapid ac id quenching as well as photometric techniques. With trypsin as target , the limiting rate constants for enzyme acylation were essentially th e same with inhibitor and substrate PAI-1 (21-23 s(-1)), as were also the associated apparent second-order rate constants (2.8-2.9 mu M-1 s( -1)). With tPA, inhibitor and substrate PAI-1 reacted identically to f orm a tightly bound Michaelis complex (K-d approximate to K-m approxim ate to 20 nM). The limiting rate constant for acylation of tPA, howeve r, was 57 times faster with inhibitor PAI-1 (3.3 s(-1)) than with the substrate form (0.059 s(-1)), resulting in a 5-fold difference in the corresponding second-order rate constants (13 vs 2.5 mu M-1 s(-1)). We attribute the ability of tPA to discriminate between the two PAI-1 fo rms to exosite bonds that cannot occur with trypsin. The exosite bonds retain specifically the distal part of the PAI-1 RCL in the substrate pocket, which favors a reversal of the acylation step. Acylation of t PA becomes effective only by separating the products of the acylation step. With substrate PAI-1, this depends on passive displacement of bo nds, whereas with inhibitor PAI-1, separation is accomplished by loop insertion that pulls tPA from its docking site on PAI-1, resulting in faster acylation than with substrate PAI-1.